This invention concerns DNA sequences that code for a glycerol-3-phosphate dehydrogenase (GPDH) and the alleles as well as the derivatives of these DNA sequences.
This invention also concerns genomic clones that contain the complete gene of a glycerol-3-phosphate dehydrogenase and alleles as well as derivatives of this gene.
This invention also concerns promoters and other regulator elements of glycerol-3-phosphate dehydrogenase genes.
Glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), also known as dihydroxyacetone phosphate reductase, is substantially involved in triacylglyceride biosynthesis in plants by supplying glycerol-3-phosphate. Fatty acid biosynthesis and triacylglyceride biosynthesis can be regarded as separate biosynthesis pathways owing to compartmentalization but as one biosynthesis pathway from the standpoint of the end product. De novo biosynthesis of fatty acids takes place in the plastids and is catalyzed by three enzymes or enzyme systems, i.e., (1) acetyl-CoA carboxylase (ACCase), (2) fatty acid synthase (FAS), and (3) acyl-[ACP]-thioesterase (TE). The end products of this reaction sequence in most organisms are either palmitic acid, stearic acid, or after desaturation, oleic acid.
In the cytoplasm, however, triacylglyceride biosynthesis takes place via the so-called "Kennedy pathway" in the endoplasmic reticulum from glycerol-3-phosphate which is made available by the activity of glycerol-3-phosphate dehydrogenase (S. A. Finnlayson et al., Arch. Biochem. Biophys., 199 (1980) pages 179-185), and from fatty acids present in the form of acyl-CoA substrates.
Probably the first discovery of the enzymatic activity of glycerol-3-phosphate dehydrogenase in plants involved potato tubers (G. T. Santora et al., Arch. Biochem. Biophys., 196 (1979) pages 403-411). This activity had not been observed in other plants before then (B. Konig and E. Heinz, Planta, 118 (1974) pages 159-169), so the existence of the enzyme had not been detected. Thus the formation of glycerol-3-phosphate on the basis of the activity of a glycerol kinase was discussed as an alternative biosynthesis pathway. Santora et al., loc. cit., subsequently detected GPDH in spinach leaves and succeeded in increasing the concentration of the enzyme approximately 10,000 times. They determined the native molecular weight to be 63.5 kDa and found the optimum pH for the reduction of dihydroxyacetone phosphate (DHAP) to be 6.8 to 9.5 for the reverse reaction. GPDH was likewise detected in Ricinus endosperm (Finlayson et al., Biochem. Biophys. 199 (1980) pages 179-185). According to more recent works (Gee et al., Plant Physiol. 86 (1988a) pages 98-103), two GPDH activities could be detected in enriched fractions, a cytoplasmic fraction (20-25%) and a plastid (75-80%) . The two forms are regulated differently. Thus, for example, the cytoplasmic isoform can be activated by F2,6DP, while the plastid isoform is activated by thioredoxin (R. W. Gee et al., Plant Physiol., 86 (1988) pages 98-103 and R. W. Gee et al., Plant Physiol., 87 (1988) pages 379-383).
The methods of molecular biology are making increasing entry into plant cultivation practice. Changes in biosynthesis output with the formation of new components and/or higher yields of these components can be achieved with the help of gene manipulation, e.g., transfer of genes which code for enzymes. As one of the most important enzymes of triacylglyceride synthesis, GPDH has a significant influence on the oil yield of plants.